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Invitro fertilization oocytes collected from preanteral and early anteral follicle of buffalo.


Buffalo is considered the animal of the future in India with its ability to survive harsher conditions as compared to cattle and also considering that much of the potential of cattle has been already exploited. In spite of being major contributor to dairy industry, only about 0.1 per cent of the milking buffaloes are capable of producing 3500-4000 kg of milk in a 305-day lactation period (Misra et al., 1990). The use of oocytes from large preantral and early antral follicles for production of embryos could offer significant new ways for the propagation of valuable animal stocks. The ovarian preantral follicles represent 90 % of the follicular population (Saumande, 1991), but the vast majority undergoes atresia during growth and maturation. Consequently, relatively few viable oocytes are produced during the reproductive life span of the female. Isolation and in vitro culture of preantral and early antral follicles aim to prevent follicular atresia by rescuing the preantral and early antral follicles population from the ovaries and by culturing the follicles in vitro up to the maturation stage and embryo production. The antral follicles on the ovarian surface are available in very limited numbers at any given time and the number available is still less in buffaloes (0.43 per one ovary; Totey et al., 1992) because of such a low germ cell reserve and high degree of follicular atresia (Danell, 1987; Bharadwaz and Roy, 1999; Rajesha et al., 2001). Also, using oocytes only from antral follicles limits the number of offspring's per animal and thus under-exploitation of the superior female genetic material (Van den Hurk et al., 1997). Since the ovaries contain a large number of preantral follicles, that can be an important alternate to surface antral follicles as a source of a good number of cultivable oocytes. Thousands of preantral follicles can be collected from an ovary using the available isolation techniques (Figueiredo et al., 1993). Ovaries from a female calf of a proven animal can even be collected to produce hundreds of superior animals utilizing the preantral follicles Buffalo preantral follicles, not much work has been done and there is still need to develop an appropriate culture system with necessary inputs and conditions for stimulating growth of preantral follicle, oocyte maturation and its gaining capacity to being fertilized in vitro. Developing such a system will not only help to produce large number embryos but also helps to understand the regulation of follicular development and atresia

IVF technology comprise of:

Oocyte (egg) collection from ovaries of slaughtered animals or using ultrasound guided aspiration from live animals

The completion of meiosis to mature the oocyte in vitro,

Preparation and treatment of spermatozoa to induce capacitation and allow fertilization,

Union of gametes to achieve in vitro fertilization and zygote formation,

Initial processing of ovaries

Ovaries collected from mature non-pregnant buffalo from a civil slaughterhouse, Bangalore were brought to the laboratory in warm (32 to 33[degrees]C) normal saline supplemented with gentamicin (50 [micro]g/ml) with in 1 hour of slaughter. Ovaries were washed 3 times in 0.9% normal saline in the laboratory and extra ovarian tissues were removed followed by washing with Dulbecco's phosphate buffer saline. Ovaries were used for the isolation of preantral, early antral follicles and collection of oocytes from visible antral follicles.

Isolation of large preantral follicles

Buffalo ovaries were brought to the laboratory in warm (32 to 33[degrees]C) normal saline supplemented with gentamicin (50 [micro]g/ml) for the isolation of preantral follicles and in ice for the isolation of somatic cells within one hour of slaughter. Ovaries were washed thoroughly in 0.9 per cent normal saline supplemented with gentamicin (50[micro]g/ml). Combined enzymatic cum microdissection method was followed for the isolation of large preantral follicles (Gupta et al., 2006: personal communication). Thin (approximately 1 mm. thick) and small sections ( approximately 5 sq. mm) were dissected out from the ovarian cortex and were washed with preantral follicle isolation medium consisting of Dulbecco's phosphate buffer saline, Steer serum (10 %) and Gentamicin (50 [micro]g/ml). The ovarian cortical sections were then digested with trypsin (1 %) by incubating at 37 [degrees]C for 10 minutes. Large preantral follicles of 150 to 500 ?m size were isolated from ovarian pieces by micro-dissection, using 26 G disposable needle and scalpel blade under stereozoom microscope. The diameter of preantral follicles was measured with precalibrated micrometer of the stereozoom microscope. Follicles with normal appearance and without visible signs of degeneration (Gupta et al., 2002a) were selected for the study.

Isolation of cumulus cells

Oocyte with cumulus cells were aspirated from surface follicle of medium sized ovarian follicles. Repeated pipetting to facilitate the release of cumulus cells that were collected and washed 3 times in culture medium before they were used and it is cultured seperately. Preantral follicles were dissected out under stereo zoom microscope and washed thrice in preservation medium consisting of TCM-199 (90 %) supplemented with follicular fluid (10 %), Mercaptoethanol (10 %) and gentamicin (50 [micro]g/ml oocytectomized follicles and were washed twice in culture medium before they were use).

Culture of preantral follicles

Preantral follicles were cultured in 100 [micro]l droplets of standard culture medium consisting of minimum essential medium (MEM) supplemented with steer serum (10 %), follicle stimulating hormone (FSH-0.05 IU/ml), sodium pyruvate (0.23Mm), glutamine (2Mm), Hypoxanthine (2Mm), Insulin-Transferrin-Selenium (ITS-6.25 [micro]g insulin, 6.25 [micro]g transferrin, 6.25ng sodium selenite), Mercaptoethanol (10 [micro]M/ml), Gentamicin (50 [micro]g/ml). Preantral follicles were cultured under mineral oil in 35 mm petridish placed in C[O.sub.2] incubator 38[degrees]C, 5 % C[O.sub.2] in air, 90-95 % relative humidity). The culture medium was replaced every alternate day. Every time, the culture medium was prepared fresh and kept for incubation in C[O.sub.2] incubator for 30 minutes before using for culture. Measuring follicle diameter on day 0 monitored the growth of follicles and thereafter every 10 days till the last day of culture. Growth rate was calculated in terms of growth achieved by the follicles in ?m per day. Vitality and morphology were assessed on days 0, 30, 60 and 80 of culture. Viability of preantral follicles was assessed by trypan blue staining technique (Gupta et al., 2002b). In this technique, follicles were treated with 0.05% (w/v) trypan blue and checked for vitality after 2 minutes. Live cells exclude the stain whereas dead cells take up the stain and appear blue in color.

Two to three preantral follicles each were co-cultured with either dispersed cumulus cells (0.7-1 X[10.sup.6] per drop of preantral follicle culture medium; group 1), or monolayer of cumulus cells (group 2)

Isolation of oocytes

After ten days follicles growth is measured and the follicles after maturation it will made to break open and the oocytes are get separated out and it is made to wash and proceed for maturation

Maturation of oocytes

In conventional in vitro maturation studies, oocytes were cultured in groups in 50-100 [micro]l droplets of TCM-199 supplemented with serum and gonadotrophins at 38.5[degrees]C in 5% C[O.sub.2] for 24 hours (Palta and Chauhan, 1998, Gupta et al., 2002) and with several other additives (Table 2). Maturation rate in vitro of oocytes are assessed by various methods like staining the oocytes (M-II stage), identification of extruded first polar body in the perivitelline space and degree of expansion of cumulus cell mass (Raghu et al., 2002). Use of chemically defined media is now recommended for oocyte culture as it negates the possible effects of unknown components of biological fluids. Different free radical scavengers like taurine, EDTA, cysteamine, melatonin, hypotaurine were being used in oocyte culture media in different species with varying results. (Funahashi et al., (1996) )has reported that the presence of organic osmolytes such as taurine and sorbital at 6 amd 12mM in maturation medium containing 68.49 or 92.40 mM NaCl increased pig oocyte glutathione content. In a study by( Harris et al. (2005)) on nutrient concentrations in murine follicular fluid have reported that taurine, glycine, alanine, glutamine and glutamate were the major amino acids detected and their concentrations differed in the follicular fluid, oviductal fluid and uterine fluid. But higher levels were found in the oviduct. In addition, The follicular fluid and tract nutrients profiles differed from those of murine maturation, fertilization and embryo culture (media.Seoka et al. (2004)) have compared the free amino acid profile with the hormonal induced Japanese eel eggs with that of corger and snake eel eggs Glutamine was the predominant amino acid in hormonal induced Japanese eel eggs, while both corger and snake eel eggs were rich in free alanine, leucine, valine, lysine ,arginine and taurine but not glutamine. Hence they had suggested that the hormonal inducement of maturity has an unfaourable effect on the free amino acid profile and quality of Japanese eel eggs. Landin et al. has carried out a study on ultrastructure of equine oocytes matured in vitro using semi-defiend culture media containing taurine. EDTA manipulates the metabolic profiles of embryos by sequestering the toxic effects of contaminating heavy metal cations and depressing glycolytic rates during early embryonic development leading to improved development (Thompson 2000). Use of cysteamine in the buffalo oocyte and embryo culture media were reported to increase the intracellular glutathione synthesis, which in turn protects the cells from oxidative stress, thus increasing the blastocyst yield (Gasparrini et al. 2001)

Culture of oocytes and the process undergoing during maturation

The cumulus cells surrounding the oocytes provide nucleotides, amino acids, phospholipids and substrates for energy utilization to the oocytes and maintain the ionic balance. Cumulus cells also protect oocytes against oxidative stress-induced apoptosis (Tatemoto et al., 2000) Oocytes originate from the primordial germ cells found in the genital ridge of fetal stage. Germ cells undergo mitosis up through 50 days and then begin undergoing meiosis until approximately 35 days after parturition. The oocytes are arrested in the dictate stage, a prolonged diplotene stage of the first meiotic division also known as the germinal vesicle stage. The primordial follicle, a single layer of squamous shaped granulosa cells, surrounds oocytes. The second phase of growth is when the follicle continues to grow while the oocyte remains arrested in dictyate stage until it reaches full adult size (>120 [micro]m) in the antral follicle. During the second growth phase, oocytes quadruple in size and depend on somatic cell (granulosa)--oocyte interactions via gap junctions. Fertilization occurs only when a spermatozoon penetrates the oocyte. In order for fertilization to occur, the spermatozoa must first undergo capacitation in which the protective plasma coating covering the spermatozoa surface molecules is removed to allow sperm to be able to bind to the oocyte. Then spermatozoa adhere to the oocytes zona pellucida to induce the acrosomal reaction, in which the fusion of the spermatozoal plasma membrane and the outer acrosomal membrane occurs to allow the acrosomal enzymes to be released. Only after these two critical events are completed can fusion of the two gametes occur.

The oocyte are surrounded by cumulus cells embedded in a thick extracellular matrix, which presents an unavoidable obstacle for spermatozoa. One of the significant components comprising the extracellular matrix is a specific disaccharide, hyaluronic acid. Spermatozoa posses an enzyme called hyaluronidase which breaks down hyaluronic acid found throughout the extracellular matrix surrounding the oocyte, thereby creating a pathway so that spermatozoa that can travel towards and adhere to the zona pellucida. A glycoprotein, PH-20, found on the head of mammalian spermatozoon has hyaluronidase activity and spermatozoa without the PH-20 on their membrane cannot traverse the cumulus cells (Lin et al., 1994). The activation of PH-20 occured during spermatozoa transport and was regulated by deglycosylation. Several new procedures involving micromanipulation are under development for assisting fertilization in vitro. Zona drilling, zona thinning and intra cytoplasmic sperm injection (ICSI) were some of them.

Statistical Analysis

The difference in size of the pre anteral and early anteral follicles observed in day 0 and different days of culture period of 30 days were tested by Dunnet test (graph pad PRISM,graph pad software inc,SanDiego,USA).Differences between the mean values were considered significant when the P value were noted

Materials and Methods

All the chemicals used in the present study were obtained from Sigma-Aldrich, USA., unless other wise stated. Trypsin and trypan blue were obtained from Himedia Labs, Mumbai, India. In the present study all the work was carried out under laminar flow (Holten Lamin air biosafety , Heto lab, Denmark).

BASIC culture Medium

Minimum essential medium has been the basic medium of choice for in vitro culture of preantral follicles in various species (Figueiredo et al., 1993; Hulshof et al., 1995; Gupta et al., 2002a). media for the in vitro culture of preantral follicles. When small preantral follicles were cultured in serum free and serum supplemented medium, the percent increase in follicular diameter after 7 days in culture was 13.6 and 28.1 per cent, respectively (Itoh and Hoshi, 2000). Bovine preantral follicle could be cultured in serum free medium. Absence of serum led to a lower increase of the follicle diameter as compared with the culture in the presence of serum. Culturing of preantral follicle with 10 per cent FBS and serum free medium resulted in a growth of 13.1 an Steer serum

Steer serum was collected by clotting the blood of a castrated bullock, heat inactivated at 56[degrees]C for 30 min., filtered (0.22mm), sterilised and stored in 2ml aliquots at--20[degrees]C until use. The same pool of serum was used throughout the study.

Assessment of the viability and growth of larger preantral and early antral follicles

The growth rate of follicles was monitored by measuring follicular diameter using a precalibrated ocular micrometer at 110x magnification on day 0 and on every 10 days of culture. Viability of preantral follicles was assessed by trypan blue staining technique (Gupta et al., 2002b). In this technique, follicles were treated with 0.05% (w/v) trypan blue and checked for vitality after 2 minutes. Live cells exclude the stain whereas dead cells take up the stain and appear blue in colour. Follicular morphology i.e. presence of dark patches within membrane granulosa and signs of degeneration for assessment of preantral follicles (Gupta et al., 2002) was assessed under stereo zoom microscope and inverted microscope (Olympus, Japan). Vitality and morphology were assessed on every 10 days of culture.

The culture medium was replaced with freshly prepared medium every alternative day. The follicular growth was monitored by measuring the follicular diameter using precalibrated ocular micro meter at 110x magnification once in 10 days

One unit of eye piece micrometry = 9[micro]after calibration

Therefore the diameter of an oocyte = number of units X 9um

And the vitality was tested once in 30 days , using trypan blue exclusion test.

Retrieval of oocytes from the cultured large preantral and early antral follicles

Oocytes were retrieved by follicle dissection method from viable follicles by using 26G needle under stereo zoom microscope and oocytes were aspirated and kept for in vitro maturation.

Grading of oocytes collected from antral follicle

Oocytes were graded by the morphological appearance of the cumulus cells and ooplasm under zoom stereomicroscope (Chauhan et al., 1998): Grade A (Good): compact oocytes with an unexpanded cumulus mass having [greater than or equal to] 5 layers of cumulus cells and with homogenous cytoplasm; grade B (fair): oocytes with 2 to 4 layers of cumulus cells with homogenous cytoplasm; Grade C (poor): oocytes without cumulus cells and with irregular (shrunken) cytoplasm (Chauhan et al., 1998). Only grade A and grade B oocytes were used for in vitro maturation

Preparation of cumulus cell monolayer

Cumulus cells @ 0.7-1 X [10.sup.6] cells were placed in 100 [micro]l droplet medium containing TCM199 + steer serum (10 %) + gentamicin (50 [micro]g/ml) and were cultured in C[O.sub.2] incubator (Forma Scientific Inc. USA) till their confluency. After 5 days of culture, the medium was replaced by preantral follicle culture medium. The preantral follicles were cultured in this medium.

In vitro maturation of oocytes

The oocytes were washed once with the aspiration medium and twice in the in vitro maturation medium (appendix) in which they would be cultured. Oocytes in groups (8 to 12 oocytes) were transferred into 50[micro]l droplets of culture medium. The droplets containing oocytes were covered with warm (38.5[degrees]C) mineral oil and the petridishes were placed in a C[O.sub.2] incubator (38.5[degrees]C, 5% C[O.sub.2] in air, 90-95% relative humidity) for 24 hours.

Assessment of oocytes for in vitro maturation

The evaluation of maturation rate of the oocytes was based on the visual assessment of the degree of expansion under zoom stereomicroscope (Kobayashi et al., 1994)): degree 0: no expansion; degree 1(moderate expansion): cumulus cells were non-homogeneously spread and clustered cells were still observed and degree 2 (fully expanded): Cumulus cells were homogeneously spread and clustered cells were no longer present. Only degree 2 and degree 1 were considered as matured. Some of the oocytes were striped off expanded cumulus cells for visualization of the extruded first polar body in the perivitelline space.

Sperm preparation and in vitro insemination

The method for processing spermatozoa for use in IVF was as standardised earlier at our laboratory (Gupta et al. 2001). Ejaculated frozen-thawed buffalo semen from 2 straws (0.25 ml, 20 million sperm cells per straw) were washed in Brackett and Oliphant (BO, Brackett and Oliphant, 1975) medium (without BSA) containing 10[micro]g/mL heparin and centrifuged twice at 500g for 5 minutes. The sperm cells were suspended for swim-up in BO medium containing 10 mg/ml heparin and 2.5 mM caffeine. Progressively motile spermatozoa were placed in 100 ml droplets of BO medium containing 0.5 per cent BSA, 10 mg/ml heparin and 2.5 mM caffeine in a petridish, covered with mineral oil, and placed in a C[O.sub.2] incubator for 1 hour at 38.5[degrees]C before inseminating in vitro-matured oocytes. The sperm concentration was then adjusted to (8-10) x [10.sup.6] /mL before inseminating the oocytes.

In vitro fertilization of oocytes

The medium in the droplets containing the matured oocytes was removed and replaced by spermatozoa (8-10) million /ml) in BO medium with 0.5 % BSA . The dishes were then placed in 5% C[O.sub.2] incubator at 38.5[degrees]C for 16 hours. After incubation period, the BO medium and unattached sperms were removed and replaced by TCM-199 supplemented by 10% steer serum along with 10-15 motile buffalo oviductal cells. The dishes were then placed again in C[O.sub.2] incubator at 38.5[degrees]C and incubated for a further period of 24 hours.

Assessment of in vitro fertilization

After 40-42 hours of insemination, presumptive zygotes were evaluated under stereo zoom microscope, for evidence of cleavage. The cleaved embryos with 2-4 cells stage or beyond were selected for in vitro culture study


A total number of 80 ovaries of buffaloes were collected from the local slaughter house . Totally 172pre antral and early antral follicles were isolated using mechanical cum enzymatic method .out of this 172 follicles 60 follicles are died due to the high concentration of mercapto ethanol .Hence the vitality percentage at the time after 20 days is 60.12%

There was a significant difference between the mean diameter of the follicle from 0 day to 20 day .It state that the maturation of follicle from day to day ..Every alter native days the media is changed and the cumulous cells separated from the oocyte is also cultured

By 30 days of in vitro culture only 60% of the follicle is survived and the follicle is made to break open and the oocytes are collected and it is put for maturation after 18-24 hours it is taken for fertilization and the cleavage is seen further stages are analyze.

Growth profile of preantral follicles during different days of in vitro culture along with cumulous cells


Growth profile of early antral follicles during different days of in vitro culture along with cumulous cells









The present study demonstrated the successful production of embryos in vitro in buffalo. The embryos were produced using oocytes collected from preantral, early antral and antral follicles. Culture of preantral follicles and early anteral folicles has important biotechnological implications through its potential to produce large qualities of oocytes for embryo production and transfer. In-vitro development system that supports oocyte growth in follicle was a difficult task because in domestic animals compared to laboratory animals, oocyte development took a long time. In present study ,an attempt was taken to culture the pre anteral and early anteral follicles along with cumulous cells to check the vitality and growth of the follicles ,oocyte retrived Combined enzymatic cum micro dissection method was followed for the isolation of large preantral follicles in buffalo (Gupta, 2006 personal communication). In this method, the ovarian cortical pieces were incubated in Trypsin of various concentrations for different periods. 1.Trypsin (1%), 37[degrees]C for 10 min. 2. Trypsin (1%), 37[degrees]C for 10 min. + 4[degrees]C for 3 hr, 3.Trypsin (0.5%), 37[degrees]C for 20 min. 4.Trypsin (0.25%), 37[degrees]C for 20 min. Further, the cortical pieces were micro dissected under stereozoom microscope.

Co culture with cumulus cells was beneficial for the development of buffalo large preantral follicles in vitro. The observations made in this study supports the findings of Wu et al. (2002) where they obtained significantly higher growth rate in preantral follicles and growth rate (%) and survival rate (%) in preantral follicular oocyte when cocultured with cumulus cells taken from large antral follicles (>3 mm). In vitro cultured cumulus cells from antral follicles (>3 mm) might secrete beneficial factors to promote in vitro growth and survival of the developing oocytes (Wu et al., 2002). Cumulus cells in antral follicles inhibited resumption of meiosis-Germinal Vesicle breakdown (GVBD) [Eppig et al., 1979; Sato et al., 1986]. The inhibitory or stimulatory effect of cumulus cells derived from antral follicles was a stage dependent process during in vitro folliculogenesis in the porcine. The CEEF induced cumulus expansion, mucification and disconnection of the gap junctions between these cells (Prochazka et al., 1991; Prochazka et al., 1998). Putative diffusible factor(s), produced by cumulus cells (CCs) and/or by the cross talk between oocyte and CCs in the intact complex, played a key role in the acquisition of developmental competence of the denuded female gamete (Luciano et al., 2005). Porcine cumulus and mural granulosa cells produce CEEF in vitro (Prochazka et al., 1998). Cumulus cells produce a diffusible meiosis-inducing substance, which overcame HX-inhibition and induced oocyte maturation, including both germinal vesicle breakdown (GVBD) and polar body (PB) formation (Guoliang et al., 1994). FSH induced cumulus cells to produce a diffusible heat stable meiosis activating substance. This substance overcame the inhibiting effect of HX and induced oocyte maturation but mural granulosa cells did not produce a meiosis inducing activity by stimulation with FSH (Byskov et al., 1997).

The recovery rate of preantral follicles was found to be significantly higher with the enzymatic method than the mechanical method. Enzymatic method yielded five times more small preantral follicles than mechanical method and the recovery rate differed significantly between the two methods and also there was no significant difference between the fresh ovaries and stored ovaries in terms of recovery rate and no significant difference between the trypsin and hyaluronidase treatment during preantral follicle separation (Gupta et al., 2001a). Combined enzymatic cum micro dissection method was followed for the isolation of large preantral follicles in buffalo (Gupta, 2006 personal communication). In this method, the ovarian cortical pieces were incubated in Trypsin of various concentrations for different periods. 1.Trypsin (1%), 37[degrees]C for 10 min. 2. Trypsin (1%), 37[degrees]C for 10 min. + 4[degrees]C for 3 hr, 3.Trypsin (0.5%), 37[degrees]C for 20 min. 4.Trypsin (0.25%), 37[degrees]C for 20 min

The first report on in vitro culture of buffaloes was made by Gupta et al (2002b) who studied the effect of FSH (0.05IU/mL) , insulin-transferrin-selenium (ITS-1%) and growth factors like EGF (50ng/mL) , FGF (50ng/mL) and VIP (50ng/mL) on the growth of buffalo preantral follicles in vitro. Small preantral follicles (diameter ranging from 36 to 72[micro]m) were cultured in groups of 2 or 4. It was demonstrated that adding both FSH and ITS individually to the culture media significantly (P<0.05) increased the follicular growth and diameter. It was found that FSH or FGF stimulate the growth of buffalo preantral follicles, and supplementation of ITS in media containing FGF significantly increased the growth rate preantral follicles. Further, stimulatory effect of EGF and VIP was found to be more pronounced in the presence of FSH than EGF alone. The study also indicated that the group culture had better influence on growth than those individual culture. Small preantral follicles in buffalo (37 to 90 [micro]m) grew faster than large preantral follicles (>91[micro]m). However very small preantral follicles (<36[micro]m) had the lowest growth rates. When the culture period was extended up to 40 days, the follicular diameter increment was significantly (P<0.05) higher in medium containing ITS + FSH and ITS + FSH + FGF and their cellular integrity was maintained. Majority of the cause for the lower cleavage rate was the quality of the frozen semen. It has been found that freezing results in acrosomal damage, leakage of enzymes, alterations in ionic strength and pH, complete withdrawal of the hydration shell of protein in the solution, and loss of motility (Meur et al. 1988). Although the fertilization rate was better in fresh semen when compared to frozen one (Totey et al. 1992) but they were less applicable due to the changes in the quality of the semen during different seasonal conditions, hence they are impractica

IU         International unit
[micro]m   Micro meter
[micro]M   Micro moles
mM         Milli moles


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Padmadhas. R., R. Ragunathan, * P.S.P. Gupta and * Sumanta Nandi

SNMV College of Arts and Science, Coimbatore, India

* National Institute of Animal Nutrition and Physiology, India

E-mail :,
Table 1: Pre anteral and early anteral follicle culture along with
cumulous cells (9-5-06)to 30-5-06)

Group                    N    Increment in size / day (diameter in
                                [micro]m) of preantral follicles

                              Day 0    Day 10    Day 20   Day 30

Pre antral follicles     12   256.45   291.23    360.15
Early antral follicles   74   540.36   569.42    631.24

Table 2: Pre antral and early antral follicles culture along with
cumulous cells(31/5/06-2/5/06)

Group                  N         % of live follicles

                            Day 0    Day 10   Day 20   Day 30

Pre antral follicles   68   243.23   274.42   345.33
Group culture          25   536.14   630.27   631.89

N: Number of preantral follicles.
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Author:Padmadhas, R.; Ragunathan, R.; Gupta, P.S.P.; Nandi, Sumanta
Publication:International Journal of Biotechnology & Biochemistry
Article Type:Report
Geographic Code:9INDI
Date:Jan 1, 2010
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